1. Field of the Invention
The present invention relates to altered antibodies in which at least part of the complementarity determining regions (CDRs) in the light or heavy chain variable domains of the antibody have been replaced by analogous parts of CDRs from an antibody of different specificity. The present invention also relates to methods for the production of such altered antibodies. The term xe2x80x9caltered antibodyxe2x80x9d is used herein to mean an antibody in which at least one residue of the amino acid sequence has been varied as compared with the sequence of a naturally occurring antibody.
2. Description of the Prior Art
Natural antibodies, or immunoglobulins, comprise two heavy chains linked together by disulphide bonds and two light chains, each light chain being linked to a respective heavy chain by disulphide bonds. The general structure of an antibody of class IgG (ie an immunoglobulin (Ig) of class gamma (G)) is shown schematically in FIG. 1 of the accompanying drawings.
Each heavy chain has at one end a variable domain followed by a number of constant domains. Each light chain has a variable domain at one end and a constant domain at its other end, the light chain variable domain being aligned with the variable domain of the heavy chain and the light chain constant domain being aligned with the first constant domain of the heavy chain. The constant domains in the light and heavy chains are not involved directly in binding the antibody to the antigen.
Each pair of light and heavy chains variable domains forms an antigen binding site. The variable domains of the light and heavy chains have the same general structure and each domain comprises four framework regions, whose sequences are relatively conserved, connected by three hypervariable or complementarity determining regions (CDRs) (see Kabat, E. A., Wu, T. T., Bilofsky, H., Reid-Miller, M. and Perry, H., in xe2x80x9cSequences of Proteins of Immunological Interestxe2x80x9d, U.S. Dept. Health and Human Services, 1983 and 1987). The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs are held in close proximity by the framework regions and, with the CDRs from the other variable domain, contribute to the formation of the antigen binding site.
For a more detailed account of the structure of variable domains, reference may be made to: Poljak, R. J., Amzel, L. M., Avey, H. P., Chen, B. L., Phizackerly, R. P. and Saul, F., PNAS USA, 70, 3305-3310, 1973; Segal, D. M., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M. and Davies, D. R., PNAS USA, 71, 4298-4302, 1974; and Marquart, M., Deisenhofers J., Huber, R. and Pale, W., J. Mol. Biol., 141, 369-391, 1980.
In recent years advances in molecular biology based on recombinant DNA techniques have provided processes for the production of a wide range of heterologous polypeptides by transformation of host cells with heterologous DNA sequences which code for the production of the desired products.
EP-A-0 088 994 (Schering Corporation) proposes the construction of recombinant DNA vectors comprising a ds DNA sequence which codes for a variable domain of a light or a heavy chain of an Ig specific for a predetermined ligand. The ds DNA sequence is provided with initiation and termination codons at its 5xe2x80x2- and 3xe2x80x2- termini respectively, but lacks any nucleotides coding for amino acids superfluous to the variable domain. The ds DNA sequence is used to transform bacterial cells. The application does not contemplate variations in the sequence of the variable domain.
EP-A-1 102 634 (Takeda Chemical Industries Limited) describes the cloning and expression in bacterial host organisms of genes coding for the whole or a part of human IgE heavy chain polypeptide, but does not contemplate variations in the sequence of the polypeptide.
EP-A-0 125 023 (Genentech Inc.) proposes the use of recombinant DNA techniques in bacterial cells to produce Igs which are analogous to those normally found in vertebrate systems and to take advantage of the gene modification techniques proposed therein to construct chimeric Igs, having amino acid sequence portions homologous to sequences from different Ig sources, or other modified forms of Ig.
The proposals set out in the above Genentech application did not lead to secretion of chimeric Igs, but these were produced as inclusion bodies and were assembled in vitro with a low yield of recovery of antigen binding activity.
The production of monoclonal antibodies was first disclosed by Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975). Such monoclonal antibodies have found widespread use not only as diagnostic reagents (see, for example, xe2x80x98Immunology for the 80sxe2x80x99, Eds. Voller, A., Bartlett, A., and Bidwell, D., MTP Press, Lancaster, 1981) but also in therapy (see, for example, Ritz, J. and Schlossman, S. F., Blood, 59, 1-11, 1982).
The recent emergence of techniques allowing the stable introduction of Ig gene DNA into myeloma cells (see, for example, Oi, V. T., Morrison, S. L., Herzenberg, L. A. and Berg, P., PNAS USA, 80, 825-829, 1983; Neuberger, M. S., EMBO J., 2, 1373-1378, 1983; and Ochi, T., Hawley, R. G., Hawley, T., Schulman, M. J., Traunecker, A., Kohler, G. and Hozumi, N., PNAS USA, 80, 6351-6355, 1983), has opened up the possibility of using in vitro mutagenesis and DNA transfection to construct recombinant Igs possessing novel properties.
However, it is known that the function of an Ig molecule is dependent on its three dimensional structure, which in turn is dependent on its primary amino acid sequence. Thus, changing the amino acid sequence of an Ig may adversely affect its activity. Moreover, a change in the DNA sequence coding for the Ig may affect the ability of the cell containing the DNA sequence to express, secrete or assemble the Ig.
It is therefore not at all clear that it will be possible to produce functional altered antibodies by recombinant DNA techniques.
However, colleagues of the present Inventor have devised a process whereby chimeric antibodies in which both parts of the protein are functional can be secreted. The process, which is disclosed in International Patent Application No. PCT/GB85/00392 (WO86/01533) (Neuberger et al. and Celltech Limited), comprises:
a) preparing a replicable expression vector including a suitable promoter operably linked to a DNA sequence comprising a first part which encodes at least the variable domain of the heavy or light chain of an Ig molecule and a second part which encodes at least part of a second protein;
b) if necessary, preparing a replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable domain of a complementary light or heavy chain respectively of an Ig molecule;
c) transforming an immortalised mammalian cell line with the or both prepared vectors; and
d) culturing said transformed cell line to produce a chimeric antibody.
The second part of the DNA sequence may encode:
i) at least part, for instance the constant domain of a heavy chain, of an Ig molecule of different species, class or subclass,
ii) at least the active portion or all of an enzyme;
iii) a protein having a known binding specificity;
iv) a protein expressed by a known gene but whose sequence, function or antigenicity is not known; or
v) a protein toxin, such a ricin.
The above Neuberger application only shows the production of chimeric antibodies in which complete variable domains are coded for by the first part of the DNA sequence. It does not show any chimeric antibodies in which the sequence of the variable domain has been altered.
EP-A-0 173 494 (The Board of Trustees of the Leland Stanford Junior University) also concerns the production of chimeric antibodies having variable domains from one mammalian source and constant domains from another mammalian source. However, there is no disclosure or suggestion of production of a chimeric antibody in which the sequence of a variable domain has been altered: indeed, hitherto variable domains have been regarded as indivisible units.
The present invention, in a first aspect, provides an altered antibody in which at least par t of a CDR in a light or heavy chain variable domain has been replaced by analogous part(s) of a CDR from an antibody of different specificity.
The determination as to what constitutes a CDR and what constitutes a framework region is made on the basis of the amino-acid sequences of a number of Igs. However, from the three dimensional structure of a number of Igs it is apparent that the antigen binding site of an Ig variable domain comprises three looped regions supported on sheet-like structures. The loop regions do not correspond exactly to the CDRs, although in general there is considerable overlap.
Moreover, not all of the amino-acid residues in the loop regions are solvent accessible and in at least one case it is known that an amino-acid residue in the framework region is involved in antigen binding. (Amit, A. G., Mariuzza, R. A., Phillips, S. E. V. and Poljak, R. J., Science, 233, 747-753, 1986).
It is also known that the variable region s of the two parts of an antigen binding site are held in the correct orientation by inter-chain non-covalent interactions. These may involve amino-acid residues within the CDRs.
Further, the three dimensional structure of CDRs, and therefore the ability to bind antigen, depends on the interaction with the framework regions: thus in some cases transplanting CDRs to a different framework might destroy antigen binding.
In order to transfer the antigen binding capacity of one variable domain to another, it may not be necessary in all cases to replace all of the CDRs with the complete CDRs from the donor variable region. It may, eg, be necessary to transfer only those residues which are accessible from the antigen binding site. In addition, in some cases it may also be necessary to alter one or more residues in the framework regions to retain antigen binding capacity: this is found to be the case with reshaped antibody to Campath 1, which is discussed below.
It may also be necessary to ensure that residues essential for inter-chain interactions are preserved in the acceptor variable domain.
Within a domain, the packing together and orientation of the two disulphide bonded beta-sheets (and therefore the ends of the CDR loops) are relatively conserved. However, small shifts in packing and orientation of these beta-sheets do occur (Lesk, A. M. and Chothia, C., J. Mol. Biol., 160, 325-342, 1982). However, the packing together and orientation of heavy and light chain variable domains is relatively conserved (Chothia, C., Novotny, J., Bruccoleri, R. and Karplus, M., J. Mol. Biol., 186, 651-653, 1985). These points will need to be borne in mind when constructing a new antigen binding site so as to ensure that packing and orientation are not altered to the deteriment of antigen binding capacity.
It is thus clear that merely by replacing at least part of one or more CDRs with complementary CDRs may not always result in a functional altered antibody. However, given the explanations set out above, it will be well within the competence of the man skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional altered antibody.
Preferably, the variable domains in both the heavy and light chains have been altered by at least partial CDR replacement and, if necessary, by partial framework region replacement and sequence changing. Although the CDRs may be derived from an antibody of the same species class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDPs will generally preferably be derived from an antibody of different species and/or from an antibody of different class or subclass.
Thus, it is envisaged, for instance, that the CDRs from a mouse antibody could be grafted onto the framework regions of a human antibody. This arrangement will be of particular use in the therapeutic use of monoclonal antibodies.
At present, if a mouse monoclonal antibody is injected into a human, the human body""s immune system recognises the antibody as foreign and produces an immune response thereto. Thus, on subsequent injections of the mouse antibody into the human, its effectiveness is considerably reduced by the action of the body""s immune system against the foreign antibody. In the altered antibody of the present invention, only the CDRs of the antibody will be foreign to the body, and this should minimise side effects if used for human therapy. Although, for example, human and mouse framework regions have characteristic sequences, to a first approximation there seem to be no characteristic features which distinguish human from mouse CDRs. Thus, an antibody comprised of mouse CDRs in a human framework may well be no more foreign to the body than a genuine human antibody.
Even with the altered antibodies of the present invention, there is likely to be an anti-idiotypic response by the recipient of the altered antibody. This response is directed to the antibody binding region of the altered antibody. It is believed that at least some anti-idiotype antibodies are directed at sites bridging the CDRs and the framework regions. It would therefore be possible to provide a panel of antibodies having the same partial or complete CDR replacements but on a series of different framework regions. Thus, once a first altered antibody became therapeutically ineffective, due to an anti-idiotype response, a second altered antibody from the series could be used, and so on, to overcome the effect of the anti-idiotype response. Thus, the useful life of the antigen-binding capacity of the altered antibodies could be extended.
Preferably, the altered antibody has the structure of a natural antibody or a fragment thereof. Thus, the altered antibody may comprise a complete antibody, an (Fabxe2x80x2)2 fragment, an Fab fragment, a light chain dimer or an Fv fragment. Alternatively, the altered antibody may be a chimeric antibody of the type described in the Neuberger application referred to above. The production of such an altered chimeric antibody can be carried out using the methods described below used in conjunction with the methods described in the Neuberger application.
The present invention, in a second aspect, comprises a method for producing an altered antibody comprising:
a) preparing a first replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least a variable domain of an Ig framework regions consisting at least parts of framework regions from a first antibody and CDRs comprising at least part of the CDRs from a second antibody of different specificity,
b) if necessary, preparing a second replicable expression vector including a suitable promoter operably linked to a DNA sequence which encodes at least the variable domain of a complementary Ig light or heavy chain respectively;
c) transforming a cell line with the first or both prepared vectors; and
d) culturing said transformed cell line to produce said altered antibody.
Preferably, the cell line which is transformed to produce the altered antibody is an immortalised mammalian cell line, which is advantageously of lymphoid origin, such as a myeloma, hybridoma, trioma or quadroma cell line. The cell line may also comprise a normal lymphoid cell, such as a B-cell, which has been immortalised by transformation with a virus, such as the Epstein-Barr virus. Most preferably, the immortalised cell line is a myeloma cell line or a derivative thereof.
Although the cell line used to produce the altered antibody is preferably a mammalian cell line, any other suitable cell line, such as a bacterial cell line or a yeast cell line, may alternatively be used. In particular, it is envisaged that E. Coli derived bacterial strains could be used.
It is known that some immortalised lymphoid cell lines, such as myeloma cell lines, in their normal state secrete isolated Ig light or heavy chains. If such a cell line is transformed with the vector prepared in step a) of the process of the invention, it will not be necessary to carry out step b) of the process, provided that the normally secreted chain is complementary to the variable domain of the Ig chain encoded by the vector prepared in step a).
In general the immortalised cell line will not secrete a complementary chain, and it will be necessary to carry out step b). This step may be carried out by further manipulating the vector produced in step a) so that this vector encodes not only the variable domain of an altered antibody light or heavy chain, but also the complementary variable domain.
Alternatively, step b) is carried out by preparing a second vector which is used to transform the immortalised cell line.
The techniques by which such vectors can be produced and used to transform the immortalised cell lines are well known in the art, and do not form any part of the invention.
In the case where the immortalised cell line secretes a complementary light or heavy chain, the transformed cell line may be produced for example by transforming a suitable bacterial cell with the vector and then fusing the bacterial cell with the immortalised cell line by spheroplast fusion. Alternatively, the DNA may be directly introduced into the immortalised cell line by electroporation. The DNA sequence encoding the altered variable domain may be prepared by oligonucleotide synthesis. This requires that at least the framework region sequence of the acceptor antibody and at least the CDRs sequences of the donor antibody are known or can be readily determined. Although determining these sequences, the synthesis of the DNA from oligonucleotides and the preparation of suitable vectors is to some extent laborious, it involves the use of known techniques which can readily be carried out by a person skilled in the art in light of the teaching given here.
If it was desired to repeat this strategy to insert a different antigen binding site, it would only require the synthesis of oligonucleotides encoding the CDRs, as the framework oligonucleotides can be re-used.
A convenient variant of this technique would involve making a synthetic gene lacking the CDRs in which the four framework regions are fused together with suitable restriction sites at the junctions. Double stranded synthetic CDR cassettes with sticky ends could then be ligated at the junctions of the framework regions. A protocol for achieving this variant is shown diagrammatically in FIG. 6 of the accompanying drawings.
Alternatively, the DNA sequence encoding the altered variable domain may be prepared by primer directed oligonucleotide site-directed mutagenesis. This technique in essence involves hybridising an oligonucleotide coding for a desired mutation with a single strand of DNA containing the region to be mutated and using the single strand as a template for extension of the oligonucleotide to produce a strand containing the mutation. This technique, in various forms, is described by: Zoller, M.e. and Smith, M., Nuc. Acids Res., 10, 6487-6500, 1982; Norris, K., Norris, F., Christainsen, L. and Ful, N., Nuc. Acids Res., 11, 5103-5112, 1983; Zoller, M. J. and Smith, M., DNA, 3, 479-488 (1984); Kramer, W., Schughart, K. and Fritz, W.-J., Nuc. Acids Res., 10, 6475-6485, 1982.
For various reasons, this technique in its simplest form does not always produce a high frequency of mutation. An improved technique for introducing both single and multiple mutations in an M13 based vector, has been described by Carter et al. (Carter, P., Bedouelle H. and Winter, G., Nuc. Acids Res., 13, 4431-4443, 1985).
Using a long oligonucleotide, it has proved possible to introduce many changes simultaneously (as in Carter et al., loc. cit.) and thus single oligonucleotides, each encoding a CDR, can be used to introduce the three CDRs from a second antibody into the framework regions of a first antibody. Not only is this technique less laborious than total gene synthesis, but is represents a particularly convenient way of expressing a variable domain of required specificity, as it can be simpler than tailoring an entire VH domain for insertion into an expression plasmid.
The oligonucleotides used for site-directed mutagenesis may be prepared by oligonucleotide synthesis or may be isolated from DNA coding for the variable domain of the second antibody by use of suitable restriction enzymes. Such long oligonucleotides will generally be at least 30 bases long and may be up to or over 80 bases in length.
The techniques set out above may also be used, where necessary, to produce the vector of part (b) of the process.
The method of the present invention is envisaged as being of particular use in reshaping human monoclonal antibodies by introducing CDRs of desired specificity. Thus, for instance, a mouse monoclonal antibody against a particular human cancer cell may be produced by techniques well known in the art. The CDRs from the mouse monoclonal antibody may then be partially or totally grated into the framework regions of a human monoclonal antibody, which is then produced in quantity by a suitable cell line. The product is thus a specifically targetted, essentially human antibody which will recognise the cancer cells, but will not itself be recognised to any significant degree, by a human""s immune system, until the anti-idiotype response eventually becomes apparent. Thus, the method and product of the present invention will be of particular use in the clinical environment.
The present invention is now described, by way of example only, with reference to the accompanying drawings.